E-coli has many uses in the field of medicine. One of the uses is for it to produce desired proteins by mixing some DNA the bacteria will implement the added DNA into their DNA sequence and now produce the desired protein for research. In order to acquire that newly made protein from the E-Coli and avoid contamination if possible, procedures are carried out in order to obtain that protein. The protein gbr-22 was expressed by allowing some bacteria to acquire that protein by heat shocking the bacteria allowing the protein to go in and mix with the DNA. The protein was centrifuged out of bacteria and purified in steps and acquiring samples of each step in order to use a SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) procedure on them. SDS-PAGE will let the user see which substances are in the solution. The SDS denatures proteins and puts a negative charge that is proportional to its mass. The negatively charged protein will attempt to move to the positively. So the heavier the protein the less it moves across the gel while the lighter the more its moves. In recent years the use of recombinant proteins in scientific research has increased greatly, as has the wealth of techniques and products used for their amplification and purification. (1) Expression, purification, and characterization techniques were able to be performed in this lab for the protein gbr22 in order to learn the process of successfully producing recombinant proteins for future labs.
Methods
This lab was taken in three parts and the first part was the protein expression in which a colony of E. Coli was mixed with gbr22 protein and another colony without in tubes. Both colonies are placed in ice for 30 minutes then were heat shocked t for 45 seconds then 2 minutes in ice. They were then given SOC media and shaken in inucbater and later placed in agar plates. Colirollers were used to spread the bacteria around. Then a single colony from each agar place was added to LB/amp media. The bacteria with gbr22 protein continued growing and the control died off. The E-coli with gbr22 protein was left overnight in a shaking incubator. The E. Coli mixed with gbr22 was centrifuged and to the pellet was added 1x PBS and lysozyme next in order for the next lab.
The next lab was the protein purification in which we purified the protein in steps until we only had the necessary protein. The solution from the previous lab was acquired and benzonase and incubated for 15 minutes room temperature. Afterwards the solution was centrifuged. The supernatant was transferred into a conical tube and the pellet was disposed off. Ni-NTA was added to the supernatant. A Bio-Rad chromatography Econo column was used to capture the supernatant mixed with the Ni-NTA. The flow was collected and labeled as waste. Imidazole was then added to the Ni-NTA resin and allowed to flow through the column again but now labeled as wash. Now from the collected wash a more concentrated form of imidazole was added and again allowed to flow through the column. Now it was labeled elution 1. 5 ml of elution buffer where placed on the column and allowed to flow now labeled as elution 2. Samples of each step where taken.
The third part is the protein characterization in which we use SDS-PAGE to separate the gbr22. First sample 1 was centrifuged and loading buffer was added to it. Samples 2-6 only had 6x gel loading buffer added to them. Then they were all placed in a heat block for 5 min at 95 C then centrifuged for 2 min at 5000 rpm. The electrophoresis tank was assembled by placing the gel cassettes in their appropriate locations and and 1x TGS buffer was added to it to fill the clamping assembly to the edge and the tank to the required level. TGS buffer was forced into the wells with a syringe to clear out debris. Now 7 ul of MW standards (Fermentas PageRuler Prestained Protein Ladder) was added to wells 2 and 9. 20 ul of each sample was added to wells 3 through 8 in their respective order. The jacks were then plugged into the power supply for 25 min at 200 V. Afterwards the gel cassettes were forcibly opened and the gel was placed in plastic containers and with nanopure water in a orbital shaker. This process was repeated three times. Then Imperial protein stain was added and mixed for 1-1.5 hours and after this water replaced the stain with a folded tissue and left overnight. The next day we a whatman filter paper and place the gel and then put saran wrap over it and place in the drying bed and lay the sealing gasket over everything.
Results
Lab Protein Expression
Figure 1A: The bacteria culture left overnight in agar plates with DNA and AMP. The DNA gave the bacteria resistance to AMP so it flourished under this environment.
Figure 1B: The bacteria culture left overnight in agar plates. This culture contained no DNA, but it did have AMP so the bacteria were terminated because of the AMP
Figure 1C: An agar plate that was coughed upon. It is strange that no bacteria cultures were found after being left overnight.
Figure 2A: After being left overnight in a shaking incubator the bacteria combined with DNA which is why they are colored
Figure 2B: The other flask left overnight in the shaking incubator also absorbed the DNA
Figure 3A: The pellet left after being centrifuged. It weighed 0.24 g
Figure 3B: The other pellet after being centrifuged weighed 0.36g
Lab Protein Purification
Figure 1: Elution 1 still has some purple in it and Elution 2 is clear of all protein.
Figure 2: Elution 1 reading at 280 wavelenght. The absorbance is 0.23.
Figure 3: Second elution 1 reading at wavelenght of 280. Absorbance is 0.22.
Fiugre 4: Elution 1 reading at maximun wavelenght of 574. Absorbance is 0.11.
Figure 5: Second elution1 reading at maximum wavelenght of 574. Absorbance is 0.25.
Protein Characterization
Figure 1: Wells 3 to 8 are the samples. Elution 1 single strand is seen on well 7 which means that the purification worked and that should be our target protein. Elution 2 is well 8 and although there should be nothing there, some protein is still faintly seen.
Figure 2: The gel broke off because the vaccuum was not turned off before the lid was lifted.
Figure 3: This is the ladder used for determing the weight of a band in the gel.
Molecular weight of gbr-22 = 25794.2 g/mol
Determining Concentration of protein using 280 nm wavelength
A=abc
A=absorbance=0.2225
a=coefficient extinction=39100(1/Mcm)
b=path length=10mm=1cm
c=concentration
c=(0.225/39100/M)=0.000006M
(0.0000006mol/L) x (1L/1000ml) x (25794.2g/mol) x (1000mg/1g)=0.148mg/ml
Determining Concentration of protein using Maximal wavelength
A=0.36
a=118300(1/Mmm)
b=1mm
c=?
c=(0.36/118300/M)=0.00003M
(0.00003/1L) x (1L/1000mL) x (25794.2g/mol) x (1000mg/g)=0.758mg/ml
Determining the yield
Maximum wavelength
(0.785mg/ml) x 4.6ml=3.5325mg
280nm wavel
ength (0.148mg/ml) x 4.5ml=0.666mg
Discussion
The only agar plate in the expression lab that had something bacteria proliferate is the gbr22 which means that the gbr22 gave the bacteria resistance to ampicillin. For the purifying lab the graphs show that at higher wavelengths the more the yield and concentration the gbr22 shows. Some errors for finding the yield for different wavelengths is using the wrong numbers in the equations which may lead to different results. For the characterization lab the gel came out looking decent since there were no significant errors by looking at the bands. Elution 1 in well 7 gave a single band and it has high intensity so I was able to characterize that specific protein out of all the other samples by purification. An error that occurred in the experiment is that when heating the gel the lid was lifted without turning off the vacuum causing the gel to break distorting several wells. Even though the wells were distorted there is only one intensive band on well 7, which is most likely the gbr22. The ladder was placed on well 2 and 9 in order to differentiate between gels since the tank was shared with other gels. Once again the SDS causes the proteins to have a negative charge so the only thing that will cause the proteins from being distinguished is the size and weight of the protein. The lighter proteins move to the bottom and the heavier are at the top. Using the protein ladder I was able to measure the molecular weight of my purified protein which is ~25 kDa. I would estimate the purity of my sample 5 to be around 99 to 100 percent since there is no band of equal intensity. Although elution 2 is not supposed to have any bands, a band is slightly visible on the gel this may be due to error when purifying; the solution was not purified well enough.
Conclusion
This lab allowed me to learn how to express, purify, and characterize a protein, which in this case was gbr22. This protein caused the bacteria to appear purple and gave them ampicillin resistance. The gel acquired after electrophoresis came out well being my first time and the purified protein appeared as a single band in well 7 meaning that there was no impurities of intense proportions that may affect the results. The procedures learned in this lab may be used in order to get a desired protein for research and acquiring that specific protein by expressing it in bacteria, purifying it, and characterizing it.
The protein gbr-22Introduction
E-coli has many uses in the field of medicine. One of the uses is for it to produce desired proteins by mixing some DNA the bacteria will implement the added DNA into their DNA sequence and now produce the desired protein for research. In order to acquire that newly made protein from the E-Coli and avoid contamination if possible, procedures are carried out in order to obtain that protein. The protein gbr-22 was expressed by allowing some bacteria to acquire that protein by heat shocking the bacteria allowing the protein to go in and mix with the DNA. The protein was centrifuged out of bacteria and purified in steps and acquiring samples of each step in order to use a SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) procedure on them. SDS-PAGE will let the user see which substances are in the solution. The SDS denatures proteins and puts a negative charge that is proportional to its mass. The negatively charged protein will attempt to move to the positively. So the heavier the protein the less it moves across the gel while the lighter the more its moves. In recent years the use of recombinant proteins in scientific research has increased greatly, as has the wealth of techniques and products used for their amplification and purification. (1) Expression, purification, and characterization techniques were able to be performed in this lab for the protein gbr22 in order to learn the process of successfully producing recombinant proteins for future labs.
Methods
This lab was taken in three parts and the first part was the protein expression in which a colony of E. Coli was mixed with gbr22 protein and another colony without in tubes. Both colonies are placed in ice for 30 minutes then were heat shocked t for 45 seconds then 2 minutes in ice. They were then given SOC media and shaken in inucbater and later placed in agar plates. Colirollers were used to spread the bacteria around. Then a single colony from each agar place was added to LB/amp media. The bacteria with gbr22 protein continued growing and the control died off. The E-coli with gbr22 protein was left overnight in a shaking incubator. The E. Coli mixed with gbr22 was centrifuged and to the pellet was added 1x PBS and lysozyme next in order for the next lab.
The next lab was the protein purification in which we purified the protein in steps until we only had the necessary protein. The solution from the previous lab was acquired and benzonase and incubated for 15 minutes room temperature. Afterwards the solution was centrifuged. The supernatant was transferred into a conical tube and the pellet was disposed off. Ni-NTA was added to the supernatant. A Bio-Rad chromatography Econo column was used to capture the supernatant mixed with the Ni-NTA. The flow was collected and labeled as waste. Imidazole was then added to the Ni-NTA resin and allowed to flow through the column again but now labeled as wash. Now from the collected wash a more concentrated form of imidazole was added and again allowed to flow through the column. Now it was labeled elution 1. 5 ml of elution buffer where placed on the column and allowed to flow now labeled as elution 2. Samples of each step where taken.
The third part is the protein characterization in which we use SDS-PAGE to separate the gbr22. First sample 1 was centrifuged and loading buffer was added to it. Samples 2-6 only had 6x gel loading buffer added to them. Then they were all placed in a heat block for 5 min at 95 C then centrifuged for 2 min at 5000 rpm. The electrophoresis tank was assembled by placing the gel cassettes in their appropriate locations and and 1x TGS buffer was added to it to fill the clamping assembly to the edge and the tank to the required level. TGS buffer was forced into the wells with a syringe to clear out debris. Now 7 ul of MW standards (Fermentas PageRuler Prestained Protein Ladder) was added to wells 2 and 9. 20 ul of each sample was added to wells 3 through 8 in their respective order. The jacks were then plugged into the power supply for 25 min at 200 V. Afterwards the gel cassettes were forcibly opened and the gel was placed in plastic containers and with nanopure water in a orbital shaker. This process was repeated three times. Then Imperial protein stain was added and mixed for 1-1.5 hours and after this water replaced the stain with a folded tissue and left overnight. The next day we a whatman filter paper and place the gel and then put saran wrap over it and place in the drying bed and lay the sealing gasket over everything.
Results
Lab Protein Expression
Lab Protein Purification
Protein Characterization
Molecular weight of gbr-22 = 25794.2 g/mol
Determining Concentration of protein using 280 nm wavelength
A=abc
A=absorbance=0.2225
a=coefficient extinction=39100(1/Mcm)
b=path length=10mm=1cm
c=concentration
c=(0.225/39100/M)=0.000006M
(0.0000006mol/L) x (1L/1000ml) x (25794.2g/mol) x (1000mg/1g)=0.148mg/ml
Determining Concentration of protein using Maximal wavelength
A=0.36
a=118300(1/Mmm)
b=1mm
c=?
c=(0.36/118300/M)=0.00003M
(0.00003/1L) x (1L/1000mL) x (25794.2g/mol) x (1000mg/g)=0.758mg/ml
Determining the yield
Maximum wavelength
(0.785mg/ml) x 4.6ml=3.5325mg
280nm wavel
ength
(0.148mg/ml) x 4.5ml=0.666mg
Discussion
The only agar plate in the expression lab that had something bacteria proliferate is the gbr22 which means that the gbr22 gave the bacteria resistance to ampicillin. For the purifying lab the graphs show that at higher wavelengths the more the yield and concentration the gbr22 shows. Some errors for finding the yield for different wavelengths is using the wrong numbers in the equations which may lead to different results. For the characterization lab the gel came out looking decent since there were no significant errors by looking at the bands. Elution 1 in well 7 gave a single band and it has high intensity so I was able to characterize that specific protein out of all the other samples by purification. An error that occurred in the experiment is that when heating the gel the lid was lifted without turning off the vacuum causing the gel to break distorting several wells. Even though the wells were distorted there is only one intensive band on well 7, which is most likely the gbr22. The ladder was placed on well 2 and 9 in order to differentiate between gels since the tank was shared with other gels. Once again the SDS causes the proteins to have a negative charge so the only thing that will cause the proteins from being distinguished is the size and weight of the protein. The lighter proteins move to the bottom and the heavier are at the top. Using the protein ladder I was able to measure the molecular weight of my purified protein which is ~25 kDa. I would estimate the purity of my sample 5 to be around 99 to 100 percent since there is no band of equal intensity. Although elution 2 is not supposed to have any bands, a band is slightly visible on the gel this may be due to error when purifying; the solution was not purified well enough.
Conclusion
This lab allowed me to learn how to express, purify, and characterize a protein, which in this case was gbr22. This protein caused the bacteria to appear purple and gave them ampicillin resistance. The gel acquired after electrophoresis came out well being my first time and the purified protein appeared as a single band in well 7 meaning that there was no impurities of intense proportions that may affect the results. The procedures learned in this lab may be used in order to get a desired protein for research and acquiring that specific protein by expressing it in bacteria, purifying it, and characterizing it.
References
1. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/index.html (accessed April 16, 2011)